Compositions and methods of use of 8-nitroguanine

ABSTRACT

Novel methods for the synthesis of 8-nitroguanine are provided. Compositions comprising 8-nitroguanine, made by the novel synthetic methods are also provided herein. Methods of use of 8-nitroguanine, made by the novel synthetic methods, as a standard for detection of 8-nitroguanine in samples are also encompassed within the scope of the present invention. The present invention further concerns methods of predicting organ transplant rejection and detecting exposure to environmental stressors, such as ionizing radiation, toxic chemicals or infectious agents, by detecting 8-nitroguanine in one or more samples from a transplant recipient or an organism exposed to stress.

This is a divisional of application Ser. No. 09/850,646, filed on May 7,2001, that has been allowed to issue. The issued patent claims thebenefit of U.S. Provisional Application No. 60/203,062 filed May 9,2000, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of surrogate markers for thedetection of stress in an organism. More specifically, the presentinvention relates to methods of use of 8-nitroguanine to detect stressin an organism, particularly as an early predictor of organ rejection ina transplant recipient. The present invention further relates to novelmethods of synthesis of 8-nitroguanine and compositions comprising8-nitroguanine.

2. Description of Related Art

Peroxynitrite is formed in the body by macrophages as part of theinflammation process (Yermilov et al., 1995a; Byun et al., 1999).Peroxynitrite can react with DNA to form 8-nitroguanine (Yermilov etal., 1995a, 1995b, 1996). It has been speculated that peroxynitrite maycause DNA or tissue damage, contributing to the multistagecarcinogenesis process (Yermilov et al., 1995a; Douki et al., 1996;Spencer et al., 1996). Formation of 8-nitroguanine as a result ofexposure to peroxynitrite could result in spontaneous depurination andsingle-stranded DNA breaks (Yermilov et al., 1995a), resulting in therelease of 8-nitroguanine. Thus, 8-nitroguanine could potentially serveas a surrogate marker for stress in general and for inflammation-relatedstress in particular. More recently, it has been suggested that8-nitroguanine does not spontaneously depurinate and that prior reportsof spontaneous depurnation were an artifact induced by non-physiologicalexposure to peroxynitrite (Tuo et al., 2000). The potential use of8-nitroguanine as a surrogate marker for inflammation and stress, priorto the present invention, was thus uncertain.

Methods for non-invasive monitoring of stress, through the detection andquantification of 8-nitroguanine in body fluids such as blood, urine andsputum would be highly desirable. Such methods could provide, forexample, a non-invasive method for predicting the likelihood of organrejection in transplant recipients, or for detecting exposure toenvironmental Stressors in the form of ionizing radiation, toxicchemicals or infectious agents like viruses and bacteria.

The development of such monitoring procedures would be facilitated bythe availability of a low-cost method for production of 8-nitroguanine,which would be of use as a standard for calibration of monitoringsystems. Present methods for the production of 8-nitroguanine areexpensive and require the use of starting materials, such as8-bromoguanine or peroxynitrite (Yermrilov et al., 1995b; Spencer etal., 1996), that are highly toxic or that may not be readily available.

SUMMARY OF THIE INVENTION

The present invention satisfies a long-standing need in the field, byproviding a novel and low-cost method for production of 8-nitroguanine.In another embodiment, the present invention concerns methods fordetecting and quantifying 8-nitroguanine in fluids, such as blood, urineor sputum. Further embodiments of the present invention concerncompositions comprising 8-nitroguanine, made by the disclosed methods.Such compositions are of use as standards for calibrating equipment todetect 8-nitroguanine in samples.

In certain embodiments, the compositions and methods of the presentinvention are used for the detection and/or quantitation of exposure toenvironmental stressors such as ionizing radiation, toxic chemicals orinfectious agents. In a preferred embodiment, the compositions andmethods are of use for predicting the likelihood of organ rejection intransplant recipients. Such a non-invasive method for predicting thelikelihood of organ rejection is superior to present methods thatrequire biopsy samples from the transplanted organ.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates the formation of 8-nitroguanine in DNA upon exposureto peroxynitrate and the β-elimination pathway for depurination andformation of single-stranded DNA breaks.

FIG. 2 illustrates alternative pathways for production of8-nitroguanine.

FIG. 3 illustrates the chromatography of 8-nitroguanine made by exposureof guanine to acetic anhydride and nitric acid.

FIG. 4 shows the absorbance profiles of peaks resulting from exposure ofguanine to acetic anhydride and nitric acid.

FIG. 5A shows the chromatogram for the products of synthetic method 3,detected at 254 nm.

FIG. 5B shows the chromatogram for the products of synthetic method 4,detected at 254 nm.

FIG. 5C shows the chromatogram for the products of synthetic method 3,detected at 380 nm.

FIG. 5D shows the chromatogram for the product of synthetic method 4,detected at 380 nm.

FIG. 6 shows the chromatogram for the products of synthetic method 5,detected at 390 nm.

FIG. 7A shows the chromatogram for the products of synthetic method 6,using dimethlyformamide as the solvent.

FIG. 7B shows the chromatogram for the products of synthetic method 6,using water as the solvent.

FIG. 8A shows the chromatogram for the products of synthetic method 4,using a long reaction time and detected at 254 nm.

FIG. 8B shows the chromatogram for the products of synthetic method 4,using a long reaction time followed by extraction with HCl and detectedat 254 nm.

FIG. 8C shows the chromatogram for the products of synthetic method 4,using a long reaction time and detected at 380 nm.

FIG. 8D shows the chromatogram for the products of synthetic method 4,using a long reaction time followed by extraction with HCl and detectedat 380 nm.

FIG. 9 shows a representative chromatogram for a urine sample from apatient with a chronic infection. A peak eluting at 4.83 minutes ismarked.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As used herein, the terms “a” and “an” mean one or more than one of anitem.

As used herein to describe acid solutions, the term “concentrated” meansthat the solution comprises at least 10%, more preferably at least 20%,more preferably at least 30%, more preferably at least 40%, morepreferably at least 50%, more preferably at least 60%, more preferablyat least 70%, more preferably at least 80%, most preferably at least 90%by weight of the acid in the solution. Calculation of weight percent ofacid solutions is well known in the art.

Methods for Low Cost Production of 8-Nitroguanine

The present invention discloses simple, efficient and low-cost methodsfor the production of 8-nitroguanine. The skilled artisan will realizethat the methods disclosed are exemplary only. Modifications of theprotocol contemplated within the scope of the invention include, but arenot limited to, changes in reaction temperature, length of reactiontime, pH of reaction, mixing conditions (e.g., stirring, refluxing,shaking), solvent, nitrating agent used and means of collecting8-nitroguanine. Such modifications are a matter of routineexperimentation for the skilled artisan, given the present disclosureand knowledge in the art of organic chemical synthesis.

The disclosed methods start with a suspension of guanine in anappropriate solvent. Exemplary solvents disclosed include acetonitrile,triflouoroacetic anhydride, water and dimethylformamide. However, othersolvents of similar characteristics, for example nitromethane, are wellknown in the art and may be substituted within the scope of theinvention.

The guanine is converted to 8-nitroguanine by addition of a nitratingagent. Exemplary nitrating agents disclosed herein include acetylnitrate, nitronium tetrafluoroborate, trifluoroacetyl nitrate, nitricacid, and sodium nitrite. The skilled artisan will realize thatalternative nitrating agents known in the art may be used in thepractice of the invention. The only requirement is that the agent usedmust nitrate guanine to form 8-nitroguanine.

In certain embodiments, the 8-nitroguanine formed may be collected fromthe solvent by a variety of means known in the art. In preferredembodiments, 8-nitroguanine is present as part or all of a precipitate.The precipitate may be easily separated from the solvent by standardtechniques. In certain preferred embodiments, the precipitate iscollected by centrifugation, followed by removal of the liquid. Thecollected precipitate may be subject to one or more wash steps, followedby repeated centrifugation and drying. Other exemplary means ofcollection include filtration, allowing the precipitate to settle anddecanting the liquid, or removing the liquid by lyophilization ordrying. The only requirement of the collection step is that the8-nitroguanine be separated from the bulk liquid component of thereaction mix. In certain embodiments where a dissolved form of8-nitroguanine is desired, it is contemplated within the scope of theinvention that collection of 8-nitroguanine may not be necessary.

Non-limiting methods for formation of 8-nitroguanine are disclosed inthe Examples section below. The skilled artisan will realize that thescope of the present invention is not limited to the preferredembodiments disclosed in the Examples. The disclosed methods aresuperior to other methods for synthesis of 8-nitroguanine, for exampleby reaction of 8-bromoguanine with sodium nitrite (Tretyakova et al.,2000), or by reacting guanine with peroxynitrite (Yermilov et al.,1995b; Tuo et al., 2000). 8-Bromoguanine is expensive and not widelyavailable, while peroxynitrite is highly toxic. The methods of thepresent invention use relatively inexpensive starting materials ofcomparatively low toxicity.

Separation and Quantitation of 8-Nitroguanine

It may be desirable to separate 8-nitroguanine from other sampleconstituents for the purposes of detection, quantitation, analysis orpurification. The skilled artisan will realize that any methods known inthe art for identification, purification or quantitation of smallorganic molecules like 8-nitroguanine may be used within the scope ofthe present invention. Examples of non-limiting techniques for8-nitroguanine analysis would include mass spectrometry, highperformance liquid chromatography (HPLC), gas chromatography, UV/VISspectroscopy, electrochemical detection and capillary electrophoresis(Yermilov et al., 1995a, 1995b, 1996; Douki et al., 1996; Spencer etal., 1996; Diplock et al., 1998; Byun et al., 1999; Tretyakova et al.,2000, Tuo et al., 2000). In a preferred embodiment, 8-nitroguanine canbe detected and quantified using aptamers (e.g., Jayasena, 1999) thatbind specifically to 8-nitroguanine. The techniques discussed below arefor exemplary purposes only and are not meant to limit the scope of theinvention to the disclosed methods.

For analysis of 8-nitroguanine from biological samples, it may bedesirable to pre-treat the sample to remove various components (wholecells, cell fragments, macromolecules, salts, precipitates) that couldinterfere with the analysis. Such methods are well known in the art andcan include sample homogenization, enzymatic digestion, detergent orsolvent extraction, centrifugation, filtration or precipitation. Foranalysis of urine samples, various contaminants may be precipitated outbefore analysis by acidification of the sample.

HPLC

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with high resolution of peaks. This is achieved by theuse of very fine particles and high pressure to maintain an adequateflow rate. Separation can be accomplished in a matter of minutes, or atmost an hour. Moreover, only a very small volume of the sample is neededbecause the particles are so small and close-packed that the void volumeis a very small fraction of the bed volume. Also, the concentration ofthe sample need not be very great because the bands are so narrow thatthere is very little dilution of the sample.

Separation of 8-nitroguanine by HPLC is well known in the art. Forexample, reverse-phase HPLC purification of 8-nitroguanine has been usedby Douki et al. (1996), Spencer et al. (1996) and Byun et al. (1999).Reverse-phase HPLC of 8-nitroguanine that had been reduced to8-aminoguanine was disclosed by Yermilov et al. (1996). Furthernon-limiting examples of HPLC analysis of 8-nitroguanine are disclosedbelow in the Examples section. A preferred method of detection of8-nitroguanine with HPLC involves the use of an electrochemical detectorassociated with the HPLC system.

Mass Spectrometry

The basis of mass spectrometry is the identification of compounds bydetermination of their molecular mass. An exemplary method for detectionof 8-nitroguanine by mass spectrometry was disclosed by Tretyakova etal. (2000), using negative-ion electrospray mass spectrometry. Otherexemplary methods of detection using mass spectrometry and gaschromatography were disclosed in Spencer et al. (1996) and Byun et al.(1999). Tuo et al. (2000) disclosed a method of detection using HPLCwith tandem mass spectrometry.

Microfluidic Techniques

Microfluidic techniques include separation on a platform such asmicrocapillaries, designed by ACLARA BioSciences Inc., or the LabChip™liquid integrated circuits made by Caliper Technologies Inc. Thesemicrofluidic platforms require only nanoliter volumes of sample, incontrast to the microliter volumes required by other separationtechnologies. Miniaturizing some of the processes involved in geneticanalysis has been achieved using microfluidic devices. For example,published PCT Application No. WO 94/05414, U.S. Pat. Nos. 5,304,487,5,296,375 and 5,856,174 each of which is incorporated herein byreference.

Capillary Electrophoresis

In some embodiments microcapillary arrays are contemplated to be usedfor the analysis of 8-nitroguanine. Microcapillary array electrophoresisgenerally involves the use of a thin capillary or channel that may ormay not be filled with a particular separation medium. Electrophoresisof a sample through the capillary provides a size based separationprofile for the sample. The use of microcapillary electrophoresis hasbeen reported in, e.g., Woolley and Mathies (1994). Microcapillary arrayelectrophoresis generally provides a rapid method for size-basedanalysis of molecules. The high surface to volume ratio of thesecapillaries allows for the application of higher electric fields acrossthe capillary without substantial thermal variation across thecapillary, consequently allowing for more rapid separations.Microfabrication of microfluidic devices including microcapillaryelectrophoretic devices has been discussed in detail (e.g., Jacobsen etal., 1994; Effenhauser et al., 1994; Harrison et al., 1993; Effenhauseret al., 1993; Manz et al., 1992; and U.S. Pat. No. 5,904,824,incorporated herein by reference. Typically, these methods comprisephotolithographic etching of micron scale channels on silica, silicon orother crystalline substrates or chips, and can be readily adapted foruse in the present invention. In some embodiments, the capillary arraysmay be fabricated from the same polymeric materials described for thefabrication of the body of the device, using injection moldingtechniques.

Tsuda et al., 1990, describes rectangular capillaries, an alternative tothe cylindrical capillary glass tubes. Some advantages of these systemsare their efficient heat dissipation due to the large height-to-widthratio and, hence, their high surface-to-volume ratio and their highdetection sensitivity for optical on-column detection modes. These flatseparation channels have the ability to perform two-dimensionalseparations, with one force being applied across the separation channel,and with the sample zones detected by the use of a multi-channel arraydetector.

In many capillary electrophoresis methods, the capillaries, e.g., fusedsilica capillaries or channels etched, machined or molded into planarsubstrates, are filled with an appropriate separation/sieving matrix.Typically, a variety of sieving matrices are known in the art may beused in the microcapillary arrays. Examples of such matrices include,e.g., hydroxyethyl cellulose, polyacrylaride, agarose and the like.Generally, the specific gel matrix, running buffers and runningconditions are selected to maximize the separation characteristics ofthe particular application. For example, running buffers may includedenaturants, chaotropic agents such as urea or the like, to denaturenucleic acids in the sample.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

Aptamers

In certain preferred embodiments, the identification and quantitation of8-nitroguanine may be accomplished using one or more aptamers thatspecifically bind to 8-nitroguanine. The term “aptamer” refers to anoligonucleotide that is capable of forming a complex with an intendedtarget substance (“analyte”), such as 8-nitroguanine. The binding istarget-specific in the sense that other materials which may accompanythe target do not bind to the aptamer. “Target-specific” means that theaptamer binds to target analyte with a much higher degree of affinitythan it binds to contaminating materials. The meaning of specificity inthis context is thus similar to the meaning of specificity as applied toantibodies, for example.

Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in Losch and Szostak (1996) and in U.S. Pat. Nos. 5,582,981,5,595,877 and 5,637,459, each incorporated herein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other aptamers specific for the same target. Further,the term “aptamer” specifically includes “secondary aptamers” containinga consensus sequence derived from comparing two or more known aptamersthat bind to a given target.

As used in this section of the specification, “binding” refers to aninteraction or binding between a target and an oligonucleotide oraptamer, resulting in a sufficiently stable complex so as to permitseparation of oligonucleotide:target complexes from uncomplexedoligonucleotides under given binding or reaction conditions. Binding ismediated through hydrogen bonding or other molecular forces.

In general, a minimum of approximately 3 nucleotides, preferably atleast 5 nucleotides, are necessary to effect specific binding. The onlyapparent limitations on the binding specificity of thetarget/oligonucleotide complexes of the invention concern sufficientsequence to be distinctive in the binding oligonucleotide and sufficientbinding capacity of the target substance to obtain the necessaryinteraction. Oligonucleotides of sequences shorter than 10 bases may befeasible if the appropriate interaction can be obtained in the contextof the environment in which the target is placed, although aptamers ofabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 nucleotides or more in length are contemplated within thescope of the present invention. Although in preferred embodiments theoligonucleotides are single-stranded or double-stranded, it iscontemplated that aptamers may sometimes assume triple-stranded orquadruple-stranded structures.

The specifically binding oligonucleotides need to contain the sequencethat confers binding specificity, but may be extended with flankingregions and otherwise derivatized. In preferred embodiments of theinvention, aptamer binding sites will be flanked by known, amplifiablesequences, facilitating the amplification of the nucleic acids by PCR orother amplification techniques. In a further embodiment, the flankingsequence may comprise a specific sequence that preferentially recognizesor binds a moiety to enhance the immobilization of the aptamer to asubstrate.

The aptamers found to bind to the targets may be isolated, sequenced,and/or amplified or synthesized as conventional DNA or RNA molecules.Alternatively, aptamers of interest may comprise modified oligomers. Anyof the hydroxyl groups ordinarily present in nucleic acids may bereplaced by phosphonate groups, phosphate groups, protected by astandard protecting group, or activated to prepare additional linkagesto other nucleotides, or may be conjugated to solid supports. The 5′terminal OH is conventionally free but may be phosphorylated. Hydroxylgroup substituents at the 3′ terminus may also be phosphorylated. Thehydroxyls may be derivatized, by standard protecting groups. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, exemplary embodiments whereinP(O)O is replaced by P(O)S, P(O)NR₂, P(O)R, P(O)OR′, CO, or CNR₂,wherein R is H or alkyl (1-20C) and R′ is alkyl (1-20C); in addition,this group may be attached to adjacent nucleotides through O or S. Notall linkages in an oligomer need to be identical.

The oligonucleotides used as starting materials in the process of theinvention to determine 8-nitroguanine specific binding sequences may besingle-stranded or double-stranded DNA or RNA. In a preferredembodiment, the sequences are single-stranded DNA The use of DNAeliminates the need for conversion of RNA aptamers to DNA by reversetranscriptase prior to PCR amplification. Furthermore, DNA is lesssusceptible to nuclease degradation than RNA. In preferred embodiments,the starting nucleic acid will contain a randomized sequence portion,generally including from about 10 to 400 nucleotides, more preferably 20to 100 nucleotides. The randomized sequence is flanked by primersequences that permit the amplification of nucleic acids found to bindto the 8-nitroguanine. The flanking sequences may also contain otherconvenient features, such as restriction sites. These primerhybridization regions generally contain 10 to 30, more preferably 15 to25, and most preferably 18 to 20, bases of known sequence.

Both the randomized portion and the primer hybridization regions of theinitial oligomer population are preferably constructed usingconventional solid phase techniques. Such techniques are well known inthe art, such methods being described, for example, in Froehler, et al.,(1986a, 1986b, 1988, 1987). Oligonucleotides may also be synthesizedusing solution phase methods such as triester synthesis, known in theart. For synthesis of the randomized regions, mixtures of nucleotides atthe positions where randomization is desired are added during synthesis.

Any degree of randomization may be employed. Some positions may berandomized by mixtures of only two or three bases rather than theconventional four. Randomized positions may alternate with those thathave been specified. Indeed, it is helpful if some portions of thecandidate randomized sequence are in fact known.

SELEX Technology

A preferred method of selecting for 8-nitroguanine specific aptamersinvolves the SELEX process. The SELEX process is described in U.S. Pat.Nos. 5,475,096, and 5,270,163, (see also WO91/19813), each specificallyincorporated by reference.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the method includes the following steps.Contacting the mixture with the target under conditions favorable forbinding. Partitioning unbound nucleic acids from those nucleic acidsthat have bound specifically to target analyte. Dissociating the nucleicacid-analyte complexes. Amplifying the nucleic acids dissociated fromthe nucleic acid-analyte complexes to yield mixture of nucleic acidsthat preferentially bind to the analyte. Reiterating the steps ofbinding, partitioning, dissociating and amplifying through as manycycles as desired to yield highly specific, nucleic acids that bind withhigh affinity to the target analyte.

In the SELEX process, a candidate mixture of nucleic acids of differingsequence is prepared. The candidate mixture generally includes regionsof fixed sequences (i.e., each of the nucleic acids contains the samesequences) and regions of randomized sequences. The fixed sequenceregions are selected to: (a) assist in the amplification steps; (b)mimic a sequence known to bind to the 8-nitroguanine; or (c) promote theformation of a given structural arrangement of the nucleic acids. Therandomized sequences may be totally randomized (i.e., the probability offinding a given base at any position being one in four) or onlypartially randomized (i.e., the probability of finding a given base atany location can be any level between 0 and 100 percent).

The candidate mixture is contacted with the 8-nitroguanine underconditions favorable for binding of 8-nitroguanine to nucleic acid. Theinteraction between the 8-nitroguanine and the nucleic acids can beconsidered as forming nucleic acid-8-nitroguanine pairs with thosenucleic acids having the highest affinity for the 8-nitroguanine.

The nucleic acids with the highest affinity for the 8-nitroguanine arepartitioned from those nucleic acids with lesser affinity. Because onlya small number of sequences (possibly only one molecule of nucleic acid)corresponding to the highest affinity nucleic acids exist in themixture, it is generally desirable to set the partitioning criteria sothat a significant amount of nucleic acids in the mixture (approximately5-50%) are retained during partitioning.

Those nucleic acids selected during partitioning as having higheraffinity for the 8-nitroguanine are amplified to create a new candidatemixture that is enriched in higher affinity nucleic acids.

By repeating the partitioning and amplifying steps, each round ofcandidate mixture contains fewer and fewer weakly binding sequences. Theaverage degree of affinity of the nucleic acids to the 8-nitroguaninewill generally increase with each cycle. The SELEX process canultimately yield a mixture containing one or a small number of nucleicacids having the highest affinity for 8-nitroguanine.

In preferred embodiments, the binding interaction between 8-nitroguanineand one or more selected aptamers is highly specific. The bindinginteraction between 8-nitroguanine and high specificity aptamers willnecessarily involve more than the standard Watson-Crick hydrogen bondformation observed between guanine and cytosine residues in vivo. It isexpected that 8-nitroguanine will form hydrogen bonds with anyoligonucleotides containing a cytosine residue. However, such aptamerswill also bind to guanine and other guanine derivatives besides8-nitroguanine. In the most preferred embodiments, the high specificityaptamer binds selectively to 8-nitroguanine in preference to guanine andother guanine derivatives. The skilled artisan can readily determine therelative binding affinity of an aptamer for 8-nitroguanine compared toguanine or other guanine derivatives. For example, 8-nitroguanine may beattached to a solid support, preferably through the use of a linkermoiety. A candidate mixture of nucleic acids may be exposed to theattached 8-nitroguanine and allowed to bind. After washing, the aptamersbound to 8-nitroguanine may be exposed to a solution containing guanine.Those aptamers that are competitively released from 8-nitroguanine bysoluble guanine will be less preferred for use in detection of8-nitroguanine, as they will also react with guanine.

Aptamers produced for SELEX may be generated on a commercially availableDNA synthesizer. The random region is produced by mixing equimolaramounts of each nitrogenous base (A,C,G, and T) at each position tocreate a large number of permutations (i.e., 4 ^(n), where “n” is theoligo chain length) in a very short segment. Thus a randomized 40 mer(40 bases long) would consist of 4³⁰ or maximally 10²⁴ differentoligonucleotides. This provides dramatically more possibilities to findhigh affinity aptamers when compared to the 10⁹ to 10¹¹ variants ofmurine antibodies produced by a single mouse. The random region isflanked by two short Polymerase Chain Reaction (PCR) primer regions toenable amplification of the small subset of oligonucleotide aptamersthat bind tightly to the target analyte.

Many RNA oligonucleotides have performed well due to their propensity toform secondary and tertiary structure “binding pockets”, but RNAsesabound in nature making RNA oligonucleotides less desirable for use.Fortunately, many single and double stranded SELEX DNA aptamers havealso demonstrated specificity and high affinity binding to theirintended targets.

Nucleic Acid Chips and Aptamer Arrays

Nucleic acid chips and aptamer array technology provide a means ofrapidly screening analytes for their ability to hybridize to apotentially large number of single stranded nucleic acid probesimmobilized on a solid substrate. In preferred embodiments, the nucleicacids are DNA. Specifically contemplated are chip-based DNA technologiessuch as those described by Hacia et al., 1996 and Shoemaker et al.,1996. These techniques involve quantitative methods for analyzing largenumbers of samples rapidly and accurately. The technology capitalizes onthe binding properties of single stranded DNA to screen samples. (Peaseet al., 1994; Fodor et al., 1993; Southern et al., 1994; Travis, 1997;Lipshutz et al., 1995; Matson et al., 1995; each of which isincorporated herein by reference.)

A nucleic acid chip or aptamer array consists of a solid substrate uponwhich an array of single stranded nucleic acid molecules have beenattached. For screening, the chip or array is contacted with a samplecontaining analyte which is allowed to bind. The degree of stringency ofbinding may be manipulated as desired by varying, for example, saltconcentration, temperature, pH and detergent content of the medium. Thechip or array is then scanned to determine which nucleic acids havebound to the analyte. Prior to the present invention, DNA chips weretypically used to bind to target DNA or RNA molecules in a sample.

A variety of DNA chip formats are described in the art, for example U.S.Pat. Nos. 5,861,242 and 5,578,832 which are expressly incorporatedherein by reference. The structure of a nucleic acid chip or arraycomprises: (1) an excitation source; (2) an array of probes; (3) asampling element; (4) a detector and (5) a signalamplification/treatment system. A chip may also include a support forimmobilizing the probe.

In particular embodiments, an aptamer may be tagged or labeled with asubstance that emits a detectable signal. The tagged or labeled speciesmay be fluorescent, phosphorescent, or luminescent, or it may emit Ramanenergy or it may absorb energy. When the aptamer binds to a targetedanalyte, such as 8-nitroguanine, a signal is generated that is detectedby the chip. The signal may then be processed in several ways, dependingon the nature of the signal.

The aptamer may be immobilized onto an integrated microchip that alsosupports a phototransducer and related detection circuitry.Alternatively, an aptamer may be immobilized onto a membrane or filterwhich is then attached to the microchip or to the detector surfaceitself.

The aptamers may be directly or indirectly immobilized onto a transducerdetection surface to ensure optimal contact and maximum detection. Theability to directly synthesize on or attach polynucleotide probes tosolid substrates is well known in the art. See U.S. Pat. Nos. 5,837,832and 5,837,860 both of which are expressly incorporated by reference. Avariety of methods have been utilized to either permanently or removablyattach the nucleic acids to the substrate. Exemplary methods aredescribed above under the section on immobilization. When immobilizedonto a substrate, the aptamers are stabilized and may be usedrepeatedly.

Within the scope of the present invention, aptamers specific for8-nitroguanine could be incorporated into an aptamer array designed torapidly screen samples, such as blood, sputum or urine, for the presenceof various compounds of interest, including 8-nitroguanine.

Prediction of Organ Rejection or Detection of Exposure to EnvironmentalStressors

8-Nitroguanine may be used as a surrogate marker for predicting thelikelihood of rejection in an organ transplant recipient. In preferredembodiments, samples obtained from a transplant recipient bynon-invasive means, such as blood or urine samples, are analyzed for8-nitroguanine by the methods of the present invention. Detection of8-nitroguanine in greater than baseline levels from a blood or urinesample of a transplant recipient is predictive of organ rejection. Suchpatients should be closely monitored and subjected to an appropriatetreatment regimen, such as antibiotic, immunosuppressant or othertherapy. Methods for prediction and treatment of organ transplantrejection are discussed in U.S. Pat. Nos. 5,484,707, 6,093,723,6,113,898, 6,133,324 and 6,200,978, each incorporated herein byreference in its entirety. A particularly high likelihood of organrejection is indicated by a large spike or sustained elevated level of8-nitroguanine in the blood or urine. Individuals showing such symptomsshould be aggressively treated to reduce the risk of organ rejection.

The term “baseline levels” means levels of 8-nitroguanine that wouldnormally be found in individuals who have not been exposed toenvironmental stressors or who are not at risk for organ rejection. Thedetermination of baseline levels of 8-nitroguanine is well within theskill in the art. The skilled artisan will realize that alternativemethods for determination of baseline levels may be employed dependingon the target population and the condition to be detected. In the caseof organ transplant recipients, an exemplary procedure would be tomeasure levels of 8-nitroguanine in the patient before and aftertransplantation, with monitoring preferable on at least a daily basisfollowing transplantation. After the patient has stabilized, monitoringmay occur at longer intervals of weekly, later monthly duration. Thefrequency of monitoring will be determined by the physician's evaluationof the patient's stability. The initial determination of “baselinelevels” will be based on the levels of 8-nitroguanine observed in thepatient preceding transplant. After the patient has stabilized followingorgan transplant, a new “baseline level” may be determined from thelevels of 8-nitroguanine observed in the stabilized patient.

Alternative methods of determining “baseline levels” are available.Levels of 8-nitroguanine may be determined in a cohort of organtransplant recipients before and after transplantation. The cohort maybe monitored for 8-nitroguanine levels following transplantation and theinformation correlated with their transplant status. Individuals whoexperience transplant rejection are informative for the levels of8-nitroguanine that are predictive of transplant rejection. The levelsof 8-nitroguanine observed in such individuals preceding transplantrejection can be determined. This information can be used to construct amore defined “baseline level” of 8-nitroguanine and to establish8-nitroguanine cutoff levels where more extensive monitoring andaggressive therapy are appropriate.

In the context of determining exposure to environmental stressors, acohort of individuals who have not been exposed to environmentalstressors may be selected and the levels of 8-nitroguanine in theirblood or urine determined. From this data, the range, mean and standarddeviation of 8-nitroguanine levels in control normal individuals may bedetermined. This in turn may be used to define the “baseline levels” of8-nitroguanine for exposure to environmental stress.

Additional cohorts of individuals with known exposure to identifiedlevels of an environmental stressor, such as toxic chemicals, ionizingradiation or infectious agents may also be selected. Levels of8-nitroguanine in such individuals may be readily determined by themethods disclosed herein. This information may be used to constructtables correlating the level of exposure to an environmental stressorwith the levels of 8-nitroguanine found in the individual. Such data mayalso take into account the length of time following exposure thatsamples are taken for determination of 8-nitroguanine. The effects ofchronic versus acute exposure to an environmental stressor on8-nitroguanine levels may also be readily determined by the skilledartisan.

The skilled artisan will realize that 8-nitroguanine may be used withinthe scope of the present invention as an indicator of exposure toenvironmental stressors, such as ionizing radiation, toxic chemicals orinfectious agents. An acute exposure to such a stressor will result in aspike in 8-nitroguanine in the blood or urine of an individual, whilechronic exposure will result in long-term elevation of 8-nitroguaninelevels. The levels of 8-nitroguanine detected are indicative of thedegree of exposure, with higher exposure resulting in higher levels of8-nitroguanine.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exposure of DNA to Peroxynitrite Results in Formation of8-Nitroguanine, Followed by Depurination and Production ofSingle-Stranded DNA Breaks

Peroxynitrite can be formed in vivo by the reaction of nitric oxide (NO)with superoxide anion (Fukuyama et al., 1997; Hurst and Lymar, 1997;Burney et al., 1999; Hughes, 1999; Love, 1999; Schwemmer et al., 2000).This extremely rapid reaction occurs in macrophages during theinflammation process. While the peroxide anion is stable, the acid form,peroxynitrous acid (pKa=6.8) is very unstable, with a half life of 1.9seconds at pH 7.4. Peroxynitrite may be synthesized in vitro by, forexample, reaction of potassium or sodium nitrite with hydrogen peroxidein acid, then immediate stabilization of the peroxynitrite formed byaddition of base such as NaOH.

The reaction between nitrite and hydrogen peroxide to form peroxynitriteis acid catalyzed, but so is the decomposition of peroxynitrite. Whennitrite and hydrogen peroxide were mixed in the presence of acid, abright yellow compound formed& Addition of base upon formation of thebright yellow color stopped both the synthetic reaction and thebreakdown reaction. The final concentration of peroxynitrite varieddepending on the timing of additions. Concentrations of peroxynitriteachieved by this method, measured by a molar extinction coefficient of1.67×10³ per mole per liter at 302 nm, varied from 90 to 134 mM.

Products reported to be formed by the attack of reactive oxygen species,such as peroxynitrite, with guanine or deoxyguanosine include xanthine,8-hydroxyguanine, 8-hydroxy-deoxyguanosine, 8-oxoguanine,7-methyl-8-hydroxyguanine, 7-hydro-8-oxo-2′-deoxyguanosine,8-oxo-7,8-dihydro-2′-deoxyguanine,4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-deoxyguanosine and8-nitroguanine (Yermilov et al., 1996; Diplock et al., 1998; Burney etal., 1999; Love, 1999; Tuo et al., 2000). It has been suggested that8-nitroguanine could be used as a marker for DNA damage induced byperoxynitrite in inflamed tissues (Yermilov et al., 1995a, 1996; Byun etal., 1999). Conversely, other reports have suggested that 8-nitroguanineis not formed in vivo by spontaneous depurination of oxidatively damagedDNA (Tuo et al., 2000).

The present results confirm that exposure of DNA to peroxynitriteresults in formation of 8-nitroguanine, followed by depurination andformation of single-stranded DNA breaks, as indicated in FIG. 1.Single-stranded DNA breaks were detected by a decrease in the degree ofsupercoiling of plasmid DNA.

As the degree of DNA damage increases, the likelihood of two singlestranded breaks occurring within a few bases on the opposite strands ofdouble-stranded DNA also increases. Double-stranded DNA breaks result ina decrease in the length of the DNA molecule, detectable as a decreasedviscosity of linear double-stranded DNA. This method was also used toassay for depurination and DNA strand breakage.

Plasmid DNA Methods and Materials

E coli strain JM109 containing plasmid DNA (pUC19) was grown in 2 ml ofM9 media to which 24 μl of 2.5 mg/ml ampicillin was added. Followingincubation for approximately 8 hours, the culture was transferred to 50ml of M9 media containing ampicillin. The following day, 25 ml of theovernight culture was used to inoculate 500 ml of M9 media containingampicillin. When the optical density at 600 nm of the bacterial culturereached between 0.6 and 0.8 O.D., 50 mg of chloramphenicol, dissolved in1 ml of absolute ethanol, was added to the culture and the culture wasincubated overnight.

The next morning, cells were harvested by centrifugation. Plasmid DNAwas isolated using a Promega Wizard MaxiPrep Kit. Cells were resuspendedin resuspension solution, and lysis buffer was added. The buffer wasneutralized after 20 min, the solution was centrifuged and thesupernatant collected. To the supernatant was added 0.5 volumes ofisopropanol, which caused the DNA to precipitate. The DNA was collectedby centrifuging and resuspended in 2 ml of TAE buffer. Wizard MaxiPrepResin DNA was then added to the DNA suspension to adsorb the DNA, andthe resin transferred to a spin column. The resin was washed and the DNAwas eluted from the column with TAE buffer by centrifugation. Afterremoving the fine particles the DNA was ready for use. Its concentrationwas measured by absorbance at 260 nm (0.2 OD units=10 μg/ml).

Plasmid DNA was analyzed by electrophoresis in TAE buffer using a 1%agarose gel. Gels were run at a constant 100 volts using bromophenolblue as the tracking dye. Ethidium bromide was added to the gel tovisualize the DNA. After an appropriate time, the DNA was visualized byillumination at 302 nm. Polaroid photographs were taken, and laterdigitized for densitometric analysis.

The Effect of pH on Formation of Single-Stranded DNA Breaks

The effect of pH on the conversion of supercoils to open circles byperoxynitrite was examined. The pH range used was from 4 to 10. Bufferswere prepared at 0.5 M strength to minimize pH changes caused by theNaOH in the peroxynitrite solution. Buffers used were citrate,cacodylate, borate, phosphate and Tris.

The final volume of each microcentrifuge tube was 40 μl. Each tubecontained 5 μl of plasmid DNA and 34 μl of buffer at either pH 4, 5, 6,7, 8, 9, or 10. Controls were run at pH 4,7, and 10. One μl ofperoxynitrite was added to each tube except controls, which received 1μl of water. The DNA was incubated at room temperature for 30 minutes.DNA was analyzed as described above.

The effect of peroxynitrite concentration on the conversion ofsupercoils to open circles was determined at pH 5, 6, 7, 8, and 9. Thefinal volume of each microcentrifuge tube was 50 μl. Each tube contained5 μl of plasmid DNA and 39 μl of 0.5 M buffer. Borate was used for thepH 7, 8 and 9 buffers, while cacodylate was used for the pH 5 and 6buffers. One tube was given 1 μl of 1/20 stock concentration ofperoxynitrite, other tubes were given either 1, 2, 4, or 6 μl of 1/10stock concentration of peroxynitrite, and the remaining tubes were given1, 2, 4, or 6 μl of the stock concentration for 9 differentconcentrations of peroxynitrite per experiment. A tenth tube acted ascontrol with no peroxynitrite added. Water was added to bring volume upto 50 μl total volume. The DNA was incubated at room temperature for 30minutes. DNA was analyzed as described above.

Viscosity Studies

Stock solutions of Sigma Calf Thymus DNA were made by dissolving DNA in0.1 M Na₂HPO₄, pH 8, 0.1 M sodium cacodylate, pH 5, 0.1 M Tris, pH 8,and 0.1 M borate, pH 8. The final volume for all viscosity experimentswas 40 ml and the DNA concentration was 420 μg/ml. Enough stock DNA wasused to provide the final concentration required (usually 20 ml and thepH adjusted with either phosphoric acid or NaOH (except for pH 5 whichused HCl). Peroxynitrite was added in 10 aliquots, and the pH maintainedby the addition of phosphoric acid (except pH 5). For experiments usingTris or borate, HCl was used to maintain the pH. The volume was broughtup to 40 ml by the addition of buffer. DNA was incubated at 37° C.Viscosities of the sample and control were taken at various times at 37°C. in a Stoney Brook Disposable Viscometer.

Preparation of Peroxynitrite

Peroxynitrite was synthesized by mixing ice cold solutions of 1.38 gNaNO₂ in 20 ml of water with 20 ml of water containing 2.27 g of 30%H₂O₂ and 0.33 ml concentrated H₂SO₄. Immediately 1.12 g NaOH in 20 ml ofH₂O (ice cold) was added. The reaction was performed on ice. Manganesedioxide was added to destroy unreacted hydrogen peroxide. The suspensionwas then filtered to collect the peroxynitrite. The concentration wasdetermined using a molar extinction coefficient at 302 nm of 1.67×10³per mole per liter. Peroxynitrite was stored frozen at −120° C.

Effect of Peroxynitrite on Supercoiled DNA

The conversion of supercoiled pUC19 plasmid DNA to open circles byperoxynitrite in vitro was examined. The effect of pH on the reaction ofperoxynitrite with DNA, holding peroxynitrite concentration constant wasexamined. Then, holding pH constant, peroxynitrite concentration wasvaried. Finally, the effect of the buffer composition on the conversionof supercoils to open circles by peroxynitrite was examined.

Peroxynitrite was effective at converting supercoiled plasmid DNA toopen circles at all pHs, even though peroxynitrite is unstable at acidicpHs. Even at the acidic pH, there was very little supercoiled DNAremaining after exposure to peroxynitrite. The lowest rate of supercoilreduction (a measure of single-stranded DNA breaks) occurred at pH 7.The amount of supercoiling remaining decreased as pH was increased from7 to 10.

The effect of pH on the concentration dependence of peroxynitritereduction of DNA supercoiling was observed. The concentration effect ofperoxynitrite was similar at pH values ranging from 5 to 9,demonstrating that pH has little effect on the reaction between DNA andperoxynitrite.

The effect of pH on DNA supercoiling at constant peroxynitriteconcentration was also observed. The maximum levels of remaining DNAsupercoiling (indicative of the minimum levels of single-stranded DNAbreaks) were observed between pH 7 and 8. A buffer effect was observedon single-strand nick formation, with lower levels of peroxynitriteinduced nicks observed in Tris-phosphate buffer with EDTA, compared withborate buffer in the absence of EDTA. Under the conditions of thisstudy, a minimum of about a 45% reduction in DNA supercoiling wasobserved following exposure to peroxynitrite.

Viscosity Data

The viscosity studies examined the effect of pH (at constantperoxynitrite concentration), the effect of increasing peroxynitriteconcentration at constant pH, and the effect of buffer on viscosity.Data was collected using a falling needle viscometer. This type ofviscometer generates a very low shear force and is suitable for use withnon-Newtonian fluids, such as DNA solutions.

As peroxynitrite exposed DNA depurinates, single strand breaksaccumulate. Eventually two single strand breaks on opposite strands mayoccur within a few nucleotides of each other. When this happens, a breakin the double stranded DNA molecule results. This shortens the DNAmolecule and reduces the viscosity of the solution. The data wasrecorded as the relative viscosity and specific viscosity of thesolution.

Viscosity decreased with time of exposure to peroxynitrite at all pHvalues, decreasing within the first two hours of peroxynitrite exposure.This data agreed with be results on plasmid supercoiling. The decreasein viscosity was dependent on the concentration of peroxynitrite used.The effects of buffer composition on viscosity reduction were similar tothose reported above for supercoiled DNA. The activation energy of thedepurination reaction was determined by Arrhenius analysis. Thecalculated activation energy was 917 Joules/mole, showing a relativelylow activation energy for formation of single-stranded DNA nicks byperoxynitrite.

These results demonstrate that exposure to peroxynitrite in vitro causessingle-stranded and double-stranded breaks in DNA, consistent with theformation of 8-nitroguanine followed by spontaneous depurination of8-nitroguanine.

Example 2 Novel Method for Synthesis of 8-Nitroguanine

There is currently no simple method for producing 8-nitroguanine.8-Nitroguanine may be synthesized by reacting guanine with peroxynitrite(Yermilov et al., 1995b). However this synthesis produces products otherthan 8-nitroguanine and has relatively low efficiency, since a largeamount of unreacted guanine remains. Further, as a suspected carcinogenand mutagen, peroxynitrite is difficult to work with. The method alsorequires an extensive workup, including repeated chromatography topurify 8-nitroguanine from the contaminants. An alternative method,reacting 8-bromoguanine with sodium nitrite (Tretyakova et al., 2000),requires the use of an expensive and not generally available startingmaterial (8-bromoguanine).

An exemplary novel method for synthesis of 8-nitroguanine within thescope of the present invention makes use of acetic anhydride as asolvent and a reactant. By adding nitric acid to the acetic anhydride,acetyl nitrate is created as the nitrating agent. The nitric acid isadded step wise to acetic anhydride solvent that already contains theguanine, providing a one-step reaction for production of 8-nitroguaninefrom guanine.

The product of this reaction was analyzed in comparison with data on thepublished spectra of 8-nitroguanine. The reaction product was reduced byzinc-HCl or sodium hydrosulfite. Under these conditions, 8-nitroguanineis reduced to form 8-aminoguanine. This reduced product was compared byHPLC and spectral analysis with 8-aminoguanine synthesized by acidhydrolysis of commercially available 8-aminoguanosine.

8-Nitroguanine Synthesis

The following methods are exemplary only and are not meant to belimiting to the scope of the present invention. The skilled artisan willrealize that the materials and reaction conditions may be varied byroutine experimentation to produce 8-nitroguanine. It is contemplatedwithin the scope of the invention that a wide variety of materials andconditions may be used, so long as 8-nitroguanine is a primary productof the reaction. “Primary product” means that 8-nitroguanine forms atleast 30%, more preferably at least 40%, more preferably at least 50%,more preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, most preferablyat least 95% of the products of the reaction.

Method 1

Suspend 1 g of guanine in 25 g of acetic anhydride. Add 0.303 ml of 90%HN03. Leave overnight at room temperature with stirring. Collect yellowliquid by either vacuum filtration or centrifugation. Boil and then coolon ice. After a precipitate forms, collect by centrifugation. Lyophilizeto dryness.

Method 2

Prepare peroxynitrite as discussed above. Add 50 mg of guanine and stirovernight at room temperature. Add acid to precipitate the reactionproduct. Collect the precipitate by centrifugation an lyophilize todryness.

Method 3

Cool 0.438 ml of acetic anhydride on ice. Add 0.303 ml of 90% nitricacid to the acetic anhydride to form acetyl nitrate. To 25 ml ofacetonitrile, add 0.5 g of guanine. Once the red color disappears fromthe acetyl nitrate, slowly add it to the guanine suspension acid refluxfor about 4 hours. Collect the precipitated reaction product bycentrifugation. Wash twice with acetonitrile and dry overnight at roomtemperature.

Method 4

Suspend 0.5 g of guanine in 25 ml of acetonitrile. Add 0.439 g. ofnitronium tetrafluoroborate and reflux for about 4 hours. After aprecipitate forms, collect by centrifuging. Wash twice withacetonitrile, then wash with water to remove any remaining nitroniumtetrafluoroborate. Lyophilize to dryness.

Method 5

Suspend 1.0 g of guanine in 8.4 ml of trifluoroacetic anhydride. Add 3aliquots of 0.1 ml of 90% nitric acid and stir overnight at roomtemperature. Collect the precipitate by centrifuging. Wash the pellettwice with 0.5 M phosphate buffer, pH 7, and then wash with water toremove the buffer. Lyophilize to dryness.

Method 6

Suspend 0.1 g of 8-bromoguanine in 10 ml of either water ordimethylformamide. Add 0.1 g of sodium nitrite and reflux for about 4hours. Collect the precipitate by centrifuging. Wash the pellet twicewith water and then lyophilize to dryness.

HPLC Chromatography

In an exemplary embodiment, 8-nitroguanine may be analyzed by gradientreverse-phase HPLC using a Phenomenex C18 Aqua column (5 micron). Thebuffer system consisted of Buffer A (20 mM ammonium formate, pH 4, 1%methanol) and Buffer B (20 mM ammonium formate, pH 4, 40% methanol),using a gradient from 100% buffer A to 20% buffer A, 80% buffer B. Thegradient may be run from about 25 to about 50 minutes.

Analysis of 8-Nitroguanine

All synthetic preparations were analyzed by HHPLC, using a C18 columnwith isocratic 0.020 M ammonium fonnate, pH 4.0, 1% methanol at a flowrate of 2 ml/min. The eluate was monitored with a diode array detectorat 254 nm. Since 8-nitroguanine is one of the products produced whenperoxynitrite reacts with guanine, the retention times of the syntheticproducts were compared to the retention times of the peaks from theperoxynitrite treatment of DNA. The spectra of the products were alsocompared with the published spectra of 8-nitroguanine.

Characterization of Synthetic Products

FIG. 2 shows alternative pathways for synthesis of 8-nitroguanine. Onepathway is to react guanine in DNA with peroxynitrite. This producesmany products, one of which is 8-nitroguanine. An alternative pathwayfor synthesis of 8-nitroguanine, described above as Method 1, involvesexposure of guanine to acetic anhydride in the presence of HNO₃. TheHPLC profile of the products of this reaction is shown in FIGS. 3A and3B. The absorbance profiles for eluting peaks are shown at 254 nm (FIG.3A) and 380 nm (FIG. 3B). At 380 nm, a single peak is observed, with aretention time of 7.98 min. At 254 nm, two additional minor peaks wereobserved at lower elution volumes.

FIG. 4 shows the absorbance profile for the products of the syntheticreaction of Method 1. Three compounds are present, corresponding to thethree peaks observed at 254 nm. The absorbance spectrum of the majorpeak eluting at 7.98 minutes is indicated by the solid line in FIG. 4.Based on its similarity with previously published absorbance profiles(Yermilov et al, 1995b; Byun et al., 1999) this peak was tentativelyidentified as 8-nitroguanine. Another compound, eluting at 4.99 minutes,showed an absorbance profile very similar to guanine, as indicated inFIG. 4. The identity of the third minor peak, eluting at 5.86 minutes,is presently unknown. These results suggested that Method 1 producedonly two reaction products, with the major component tentativelyidentified as 8-nitroguanine.

Although the major peak from Method 1 showed an absorbance profilesimilar to 8-nitroguanine, these results were not confirmed by massspectrometry analysis. The molecular weight of 9-nitroguanine should be197. The molecular weight of the major product of Method 1 wasdetermined by mass spectrometry to be 198, the same as 8-nitroxanthine.It is thus believed that the major reaction product of Method 1 was8-nitroxanthine.

Method 3 was a modification of Method 1, performing the reaction inacetonitrile instead of acetic anhydride as the solvent. Method 4 was amodification of Method 3, using nitronium tetrafluoroborate as thenitrating agent in acetonitrile solvent, without any acid addition.

FIG. 5 shows the HPLC chromatograms for the reaction products of Methods3 and 4. FIG. 5A shows the reaction products of Method 3 at 254 nm. FIG.5B shows the reaction products of Method 4 at 254 nm. FIGS. 5C and 5Dshow, respectively, the reaction products of Methods 3 and 4 at 380 nm.Both Methods 3 and 4 resulted in peaks apparently corresponding to8-nitroguanine, which was a major peak in both chromatograms absorbingat 380 nm. However, Method 3 also resulted in several late eluting peaksof unknown identity.

Mass spectrometry was performed on the putative 9-nitroguanine peaksobtained from Method 3 and Method 4. Both compounds showed a molecularweight of 197, confirming the identification of the peak as8-nitroguanine. Although 8-nitroguanine was formed by these protocols,the absorbance spectra at 254 nm indicates that the reaction was notparticularly efficient.

In Method 5 the acetic anhydride of Method 1 was replaced withtrifluoroacetic anhydride. FIG. 6 shows an HPCL chromatogram for theproducts of Method 5. One major peak at 380 nm was detected, with aretention time almost identical to the 8-nitroguanine produced byMethods 3 and 4. The absorbance profile of the Method 5 peak was alsovery similar to 8-nitroguanine (not shown). Contaminating peaks werealmost negligible at 380 nm (FIG. 6).

Method 6 showed by comparison the results of reaction of 8-bromoguaninewith sodium nitrite, using either water or dimethylformamide as asolvent. The HPLC chromatograms for the products of Method 6 are shownin FIG. 7, with either dimethlyformamide (FIG. 7A) or water (FIG. 7B) asthe solvent. While a peak corresponding to 8-nitroguanine can be foundin both the water and DMF reactions, there were numerous contaminatingpeaks as well.

Method 4 was further improved in an attempt to obtain a pure preparationof 8-nitroguanine. The improved Method 4 continued to use acetonitrileas solvent, but the molar ratio of nitronium tetrafluoroborate toguanine was increased from 1:1 to 2:1 and the mixture was refluxed for12 hours. After 12 hours of refluxing, a yellow precipitate had formedin the flask. This precipitate was further purified by boiling in 1 MHCl, resulting in an extracted precipitate consisting of material thatwas insoluble in hot HCl. The HPLC chromatograms for the reactionproducts of improved Method 4 are shown in FIG. 8. FIG. 8A shows thechromatogram at 254 nm using the long reaction time Method 4. FIG. 8Bshows the chromatogram at 254 nm of the HCl extracted precipitate. FIG.8C shows the chromatogram at 380 nm of the long reaction time Method 4.FIG. 8D shows the HCl extracted precipitate at 380 nm. It is apparentfrom FIGS. 8B and 8D that the acid extracted precipitate contained analmost pure preparation of 8-nitroguanine.

In summary, the improved method 4 appears to give an almost purepreparation of 8-nitroguanine.

Example 3 Detection of 8-Nitroguanine in Urine Samples

In certain embodiments, 8-nitroguanine is detected in samples,preferably urine or blood samples, from organ transplant recipients orindividuals exposed to environmental stress, such as infection orionizing radiation. The present example demonstrates that 8-nitroguanineis detectable in urine samples from an individual with a chronicinfection.

Sample Treatment

Fresh urine samples were collected and stored on ice prior to use. Thesamples were split into four 5 ml aliquots. Samples were treated byaddition of 0.5 ml of concentrated hydrochloric acid or 0.5 ml of 37%ammonia. The pH of the acidified sample was less than 1.0. Certainsamples were spiked with 8-nitroguanine as an internal control. Additionof acid or base to urine frequently resulted in the formation of aprecipitate. Methylene chloride (5 ml) was added to removeorganic-soluble contaminants and the tubes were vigorously shaken. Sometubes were also treated by addition of 1 ml of saturated NaCl solutionbefore shaking. Samples were immediately centrifuged at 2,500 rpm for 20min in a clinical centrifuge to separate the organic and aqueous phases.The organic phase was discarded and the aqueous phase was collected foranalysis. Before HPLC chromatography, the pH was adjusted to 4.0 andmethanol was added to 4% (vol/vol). Samples were filtered through 0.2 μmnitrocellulose syringe-tip filters before injection of 0.1 ml aliquotsinto the HPLC.

Detection of 8-Nitroguanine

Samples were analyzed by reverse-phase HPLC, as described above, withthe following changes. A C18 Medichem (Woodridge, Ill.) reverse-phasecolumn was used, resulting in a decrease in total run time to 37 min.The run conditions used were: 0 to 18 min (20 mM ammonium formate, pH4.0, 4% methanol); 18 to 25 min (20 mM ammonium formate, pH 4.0, 30%methanol) and 25 to 37 min (20 mM ammonium formate, pH 4.0, 4%methanol). Eluting peaks were detected using an ESA CoulArrayelectrochemical coulometric array detector (Chelmsford, Mass.). Theelectrochemical detector was more sensitive to 8-nitroguanine than UVdetectors.

Results

FIG. 9 shows the results of HPLC analysis of a representative urinesample from a patient with a chronic infection. The samples was treatedwith base (ammonia), then neutralized to pH 4.0 as disclosed above. Apeak identified in FIG. 9 as eluting at 4.83 min corresponded to theelution time of the 8-nitroguanine internal standard (not shown). Theseresults show that 8-nitroguanine can be detected in the urine of apatient with chronic infection, corresponding to an environmentalstress.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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U.S. Pat. No. 6,133,324

U.S. Pat. No. 6,200,978

Woolley and Mathies, Proc Natl Acad Sci U S A, 91:11348-52, 1994

Yermilov, V., Rubio J., and Ohshima, H., “Formation of 8-nitroguanine inDNA treated with peroxynitrite in vitro and its rapid removal from DNAby depurination.” FEBS Letters, 376:207-210, 1995a

Yermilov, V., Rubio J., Becchi, M., Friesen, M. D., Pignatelli, B., andObshima, H., “Formation of 8-nitroguanine by the reaction of Guaninewith peroxynitrite in vitro,” Carcinogenesis 16:2045-2050, 1995b.

Yermilov, V., Yoshie, Y., Rubio J., and Ohshima, H., “Effects of carbondioxide/bicarbonate on induction of DNA single-strand breaks andformation of 8-nitroguanine, 8-oxoguanine and base-propenal mediated byperoxynitrite,” FEBS Letters, 399:67-70, 1996.

What is claimed is:
 1. A method for producing 8-nitroguanine,comprising: a) obtaining a suspension of guanine in acetonitrile; b)obtaining a solution of acetyl nitrate; c) adding the acetyl nitrate tothe guanine suspension; d) reacting the acetyl nitrate with the guanineto form 8-nitroguanine; and e) collecting the 8-nitroguanine.
 2. Themethod of claim 1, wherein the acetyl nitrate is obtained by a processcomprising, (i) cooling acetic anhydride on ice; and (ii) addingconcentrated nitric acid to the acetic anhydride to form acetyl nitrate.3. The method of claim 1, wherein the reacting step comprises refluxingfor about 4 hours.
 4. The method of claim 1, wherein the collecting stepcomprises centrifugation of a precipitate containing 8-nitroguanine. 5.A method for producing 8-nitroguanine, comprising: a) obtaining asuspension of guanine in acetonitrile; b) adding nitroniumtetrafluoroborate to the guanine suspension; c) reacting the nitroniumtetrafluoroborate with the guanine to form 8-nitroguanine; and d)collecting the 8-nitroguanine.
 6. The method of claim 5, wherein thecollecting step comprises centrifugation of a precipitate containing8-nitroguanine.
 7. The method of claim 6, wherein the collectedprecipitate is washed to remove remaining nitronium tetrafluoroborate.8. The method for producing 8-nitroguanine, comprising: a) obtaining asuspension of guanine in trifluoroacetic anhydride; b) addingconcentrated nitric acid to the guanine suspension to formtrifluoroacetyl nitrate; c) reacting the trifluoroacetyl nitrate withthe guanine to form 8-nitroguanine; and d) collecting the8-nitroguanine.
 9. The method of claim 8, wherein the reacting stepcomprises stirring overnight at room temperature.
 10. The method ofclaim 8, wherein the collecting step comprises centrifugation of aprecipitate containing 8-nitroguanine.
 11. The method of claim 10,further comprising: (i) washing the collected precipitate with buffer ata pH of about 7.0; and (ii) washing the collected precipitate with waterto remove the buffer.
 12. A method of producing 8-nitroguanine,comprising: a) obtaining a suspension of guanine in water ordimethylformamide; b) adding sodium nitrite to the guanine suspension;c) reacting the sodium nitrite with the guanine to form 8-nitroguanine;and d) collecting the 8-nitroguanine.
 13. A method for producing8-nitroguanine, comprising: a) obtaining a suspension of guanine innitromethane; b) adding nitronium tetrafluoroborate to the guaninesuspension; c) reacting the nitronium tetrafluoroborate with the guanineto for 8-nitroguanine; and d) collecting the 8-nitroguanine.
 14. Themethod of claim 5, wherein said reacting comprises refluxing for about 4hours.
 15. The method of claim 5, wherein the molar ratio of nitroniumtetrafluoroborate to guanine is 2:1 and wherein said reacting comprisesrefluxing for about 12 hours.
 16. The method of claim 5, furthercomprising boiling the collected 8-nitroguanine in 1 N HCl.
 17. Themethod of claim 16, further comprising washing the 8-nitroguanine. 18.The method of claim 12, wherein said reacting comprises refluxing forabout 4 hours.
 19. The method of claim 12, wherein said collectingcomprises centrifugation of a precipitate containing 8-nitroguanine.